Project Summary Human studies have shown that air pollution is associated with cardiovascular morbidity, yet the role of nutrition in modifying this susceptibility is unclear. Vitamin D deficiency (VDD) has become a public health concern and the adverse consequences of its interaction with other stressors needs to be understood. VDD is linked to low levels of klotho, an anti-aging protein with a critical role in sinoatrial function. Thus, I hypothesize that VDD worsens cardiovascular dysfunction triggered by air pollution exposure, especially arrhythmia, due to lower klotho expression. Our previous data show that air pollution-induced cardiovascular effects are mediated by transient receptor potential (TRP) irritant channels, specifically TRPA1, which are regulated by klotho. Thus, I surmise that worsening of these effects by VDD is due to klotho- induced changes in TRPA1 function. First, I will determine the effect of early-life VDD on cardiovascular responses to photochemical smog in mice and evaluate if VDD results in epigenetic changes that silence klotho expression. Three-week-old mice will be placed on either a VDD or normal diet for 16 weeks. Mice will either be surgically implanted with biopotential radiotelemeters to continuously measure electrocardiogram (ECG) and heart rate, or will undergo echocardiography (Echo) and whole-body plethysmography (WBP) throughout the diet, before and after smog exposure to track cardiac and ventilatory function, respectively. Mice will then be exposed to either a complex smog mixture or filtered air. ECG will be analyzed for intervals, arrhythmias and heart rate variability (HRV). Second, the DNA methylation state of klotho and gene expression of klotho and TRPA1 will also be assessed. Lastly, we will investigate if TRPA1 modulation by klotho plays a role in VDD-induced worsening of cardiovascular responses to air pollution. The involvement of TRPA1 will be determined by exposing mice to acrolein, which is a TRPA1 agonist. Mice will be treated with recombinant klotho or saline for 3 weeks at the end of the previously described VDD diet regimen to determine if the potentiated effects are blocked. ECG, HRV, Echo and WBP will be done as assessments of function. This project will be the first to characterize the role of VDD in worsening cardiovascular responses to air pollution. This work will also clarify the mechanism by which early-life VDD ?sensitizes? the cardiovascular system to air pollution-induced dysfunction.